Figure 2.
N-Rok::vhhGFP4 is a functional enzyme and efficiently phosphorylates Sqh::GFP in vivo in a tissue-specific manner. (a) Expression of N-Rok::vhhGFP4 efficiently recruits Sqh::GFP at the cell cortex in interphasic cells. Stable S2 cell line expressing Sqh::GFP, transfected with N-Rok::vhhGFP4-HA, N-RokDead::vhhGFP4-HA, N-Rok-HA, or N-RokDead-HA. Sqh::GFP is shown in green, anti-HA staining in red (transfected cells), DAPI in dark blue. Graphs on the right show the ratio of cortical Sqh::GFP/cytoplasmic Sqh::GFP of a representative cell shown for each genotype. Scale bars are 5 µm. (b) Quantification of the data shown in a. n = 218 cells, data from three independent experiments. Bars indicate mean ± SD. Asterisks denote statistical significance, derived from unpaired and two-sided Mann–Whitney tests since normality tests showed non-normal distributions: ∗∗∗∗, P ≤ 0.0001; ∗∗, P ≤ 0.01; and n.s., not significant. (c) Stable S2 cell line expressing Sqh::GFP transfected with N-Rok::vhhGFP4-HA and stained with anti-Sqh2P antibody. The right panel shows the magnification of the indicated cell region, and the white arrow points to the enrichment of phospho-Sqh signal at the cell cortex. Scale bars are 5 µm. (d) Panels show lateral views of fixed Drosophila embryos at stage 13–14 (dorsal closure) expressing Sqh::GFP and the synthetic kinase in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with anti-phospho-Sqh/MRLC antibody. The right panel shows magnification of the respective Sqh::GFP and p-Sqh images for each of the embryo genotypes shown on the left. White arrows point to the actomyosin cable around the dorsal hole, yellow arrows point to Sqh::GFP and p-Sqh foci. Please note that the “control” image is the same as in Fig. 4 a to have a single reference image for all N-Rok variants used. For every expressed synthetic kinase, the number of considered embryos is indicated (n). For N-Rok::vhhGFP4 the n number is given as a proportion of embryos in which clear p-Sqh clumps corresponding to Sqh::GFP foci were clearly visible to the total number of embryos included in the analysis. Scale bar, 20 μm; in the zoomed panel, 10 μm.

N-Rok::vhhGFP4 is a functional enzyme and efficiently phosphorylates Sqh::GFP in vivo in a tissue-specific manner. (a) Expression of N-Rok::vhhGFP4 efficiently recruits Sqh::GFP at the cell cortex in interphasic cells. Stable S2 cell line expressing Sqh::GFP, transfected with N-Rok::vhhGFP4-HA, N-RokDead::vhhGFP4-HA, N-Rok-HA, or N-RokDead-HA. Sqh::GFP is shown in green, anti-HA staining in red (transfected cells), DAPI in dark blue. Graphs on the right show the ratio of cortical Sqh::GFP/cytoplasmic Sqh::GFP of a representative cell shown for each genotype. Scale bars are 5 µm. (b) Quantification of the data shown in a. n = 218 cells, data from three independent experiments. Bars indicate mean ± SD. Asterisks denote statistical significance, derived from unpaired and two-sided Mann–Whitney tests since normality tests showed non-normal distributions: ∗∗∗∗, P ≤ 0.0001; ∗∗, P ≤ 0.01; and n.s., not significant. (c) Stable S2 cell line expressing Sqh::GFP transfected with N-Rok::vhhGFP4-HA and stained with anti-Sqh2P antibody. The right panel shows the magnification of the indicated cell region, and the white arrow points to the enrichment of phospho-Sqh signal at the cell cortex. Scale bars are 5 µm. (d) Panels show lateral views of fixed Drosophila embryos at stage 13–14 (dorsal closure) expressing Sqh::GFP and the synthetic kinase in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with anti-phospho-Sqh/MRLC antibody. The right panel shows magnification of the respective Sqh::GFP and p-Sqh images for each of the embryo genotypes shown on the left. White arrows point to the actomyosin cable around the dorsal hole, yellow arrows point to Sqh::GFP and p-Sqh foci. Please note that the “control” image is the same as in Fig. 4 a to have a single reference image for all N-Rok variants used. For every expressed synthetic kinase, the number of considered embryos is indicated (n). For N-Rok::vhhGFP4 the n number is given as a proportion of embryos in which clear p-Sqh clumps corresponding to Sqh::GFP foci were clearly visible to the total number of embryos included in the analysis. Scale bar, 20 μm; in the zoomed panel, 10 μm.

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